Two Consecutive Invasive Surgeries Utilizing Zymogen Protein C (ZPC) That Enhanced Patient Safety and Reduced Costs.

This case report describes a major surgical procedure for a protein C-deficient, hypercoagulable patient who underwent two back-to-back invasive surgeries, hip replacement, and spinal stenosis correction. The patient, an 84-year-old male with a history of deep vein thromboses (DVT) and pulmonary emboli (PE), was treated pre-, peri-, and postoperatively with zymogen protein C (ZPC-Baxter, International) and recovered without clotting or increased bleeding. During the procedure, the patient was not administered any other anticoagulants. There have now been several case reports on different patients with unrelated teams in various locations worldwide using zymogen protein C during surgical procedures. Thus, this procedure is becoming a viable choice for patients with a high probability of clotting during and after invasive surgery. This case focuses on accomplishing safer surgery and reducing costs, by using less ZPC while accomplishing two surgeries in one procedure. As a result, this procedure might be useful for many medical situations where acquired protein C deficiency could be a problem (e.g., sepsis, pregnancy, etc.). This approach may have greater application to medical conditions other than protein C deficiency, where clotting and inflammation can become issues.

A novel diagnostic algorithm for heparin-induced thrombocytopenia.

INTRODUCTION: While diagnostic algorithm using PF4-heparin enzyme-linked immunosorbent assay (ELISA) optical density (OD), and heparin neutralization assay (HNA), or 4T score have been proposed to replace serotonin-release assay (SRA) for heparin-induced thrombocytopenia (HIT), their performance against SRA is unclear. In this study, we proposed and validated the performance of a new algorithm combining PF4-heparin ELISA optical density (OD), HNA and 4T score against SRA for HIT diagnosis. METHODS: Heparin neutralization assays were performed on specimens submitted for HIT testing with positive PF4-heparin ELISA from December 2015 to September 2017, which were separated into a "training" and a "validation" data set. 4T scores were calculated for ELISA positive cases. RESULTS: A total of 123 consecutive unique patient samples had positive PF4-heparin ELISA with also HNA data, SRA data, and 4T scores available. Compared to SRA, a "laboratory criteria" (ELISA OD ≥ 1.4 and HNA ≥ 70%) had a sensitivity of 88% (14/16) and specificity of 91% (42/46), and a "combined criteria" (4T score = 8, or 4T score = 6 or 7 and ELISA OD ≥ 1.0, or 4T score = 4 or 5 and ELISA OD ≥ 2.0) had a sensitivity of 75% (12/16) and specificity of 98% (45/46) in the training data set. Laboratory and combined criteria had 90% (56/62) concordance rate. Importantly, for these concordant cases, the diagnostic specificity is 100% (46/46). Based on the data, a novel diagnostic algorithm combining these 2 criteria was proposed and validated prospectively. CONCLUSION: A novel algorithm has high diagnostic accuracy and potentially could eliminate the need for SRA testing in at least 90% patients with suspected HIT.

A Compelling Case for the Use of Perioperative Zymogen Protein C for Increased Patient Safety.

It is imperative to maintain normal blood flow to provide adequate oxygen supply to specific organs and cells, as well as for the removal of metabolic byproducts. Therefore, any situation that results in blood clotting can injure or kill living tissues. In this paper, we describe a case where a protein C deficient subject who would, by all medical indicators, be at 100 % risk of experiencing thrombophlebitis, deep vein thrombosis, and or lung emboli, is able to escape all pathologies by using perioperative zymogen protein C (ZPC). This protein C deficient patient has a long history of blood clotting, particularly from surgical procedures. The patient is 81 years old and first experienced clotting due to hernia surgery in 1964, when he was hospitalized for 16 days post-surgery with life threatening complications. It was later determined in 1980, after many episodes, that the patient had hereditary protein C deficiency at the 38 % level. In his hernia surgery, perioperative ZPC was used along with accepted anticoagulation procedures with no blood clots or other related side effects occurring. This procedure can greatly benefit protein C deficient patients, and could potentially find use for non-PC deficient patients in surgeries and a variety of other medical treatments. This particular case helps to validate the importance of ZPC in effecting safer surgery in high-risk patients. It also supports the mechanism of ZPC acting as an anticoagulant without causing bleeding. Most importantly, each clinical case study represents a unique combination of surgeon, hematologist, medical staff, and patient functioning as a coordinated team. In this case, smaller amounts of very expensive ZPC achieved safe and efficacious results, which is hugely important for future clinical applications when considering the production cost of ZPC. More studies must be done to establish minimum dosing while achieving safe and efficacious outcomes.

Retrospective cohort study comparing activated partial thromboplastin time versus anti-factor Xa activity nomograms for therapeutic unfractionated heparin monitoring in pediatrics.

BACKGROUND: Unfractionated heparin (UFH) is widely used to treat thromboembolic disease, but monitoring in children is challenging. Both activated partial thromboplastin time (aPTT) and anti-factor Xa activity (anti-Xa) are utilized, but a comparison of dosing nomograms has not been reported in pediatrics. OBJECTIVE: To compare the performance of aPTT and anti-Xa for UFH monitoring in pediatric patients. DESIGN/METHODS: A retrospective cohort study was conducted in patients ≤ 21 years old treated with UFH at Johns Hopkins Hospital from January 2009 to May 2011. For monitoring, an aPTT nomogram was used for the initial 15 months, and an anti-Xa nomogram was used for the subsequent 12 months. Clinical characteristics, laboratory data and outcomes were analyzed. RESULTS: Thirty-four patients were monitored with aPTT and 26 patients with anti-Xa. There was no significant difference in median time to therapeutic range (11.6 h aPTT, 95%CI = 6.0-17.0; 9.9 h anti-Xa, 95%CI = 7.3-20.7) or per cent of patients achieving therapeutic measurements at 24 (79% aPTT, 95%CI = 62-91; 73% anti-Xa, 95%CI = 52-88) and 48 h (88% aPTT, 95%CI = 73-97; 96% anti-Xa, 95%CI = 80-100). However, anti-Xa measurements were more frequently therapeutic than aPTT (74% [95%CI = 69-78] vs. 54% [95%CI = 50-59]). Variance between anti-Xa and aPTT measurements was high (R(2)  = 0.236). No significant difference was seen in bleeding incidence (9% aPTT, 95%CI = 2-24; 15% anti-Xa, 95%CI = 4-35). CONCLUSION: The time to achieve therapeutic measures and bleeding outcomes were not significantly different between anti-Xa and aPTT nomograms. However, a small study size limits the power to detect clinically relevant differences. The results warrant larger prospective studies of UFH monitoring in children with thromboembolic disease.

Venous thromboembolism incidence in the Cooperative Study of Sickle Cell Disease.

BACKGROUND: Venous thromboembolism (VTE) has been recently recognized as a complication of sickle cell disease (SCD); however, the incidence of VTE in SCD is unknown. OBJECTIVES: The primary objective of this study was to determine the incidence of first VTE, including pulmonary embolism (PE) and deep vein thrombosis (DVT), among SCD patients age ≥ 15 years. We also evaluated genotypic differences in VTE risk and determined the relationship between VTE and mortality. PATIENTS/METHODS: In this retrospective cohort study, we used data from the Cooperative Study of Sickle Cell Disease (CSSCD) to calculate incidence rates for first VTE. We used Cox proportional hazard models to estimate hazard ratios (HRs) for time to VTE by genotype and time to death by VTE status. RESULTS: We included 1523 SCD patients aged ≥ 15 years with 8862 years of follow-up in this analysis. The incidence rate for first VTE was 5.2 events/1000 person-years (95% confidence interval [CI] 3.8-6.9) with a cumulative incidence of 11.3% (95% CI 8.3-15.3) by age 40 years. Individuals with the SS/Sß(0) -thalassemia genotype had the highest rate of VTE (7.6 events/1000 person-years [95% CI 5.3-10.6]). The incidence of PE exceeded that of isolated DVT (3.6 [95% CI 2.5-5.1] events/1000 person-years vs. 1.6 [95% CI 0.9-2.7] events/1000 person-years), although this difference was not statistically significant. SCD patients with VTE had a higher mortality rate (adjusted HR 2.32 [95% CI 1.20-4.46]) than those without VTE. CONCLUSIONS: Patients with SCD are at substantial risk for VTE, and individuals with VTE are at higher risk of death than those without VTE.

Eculizumab prevents recurrent antiphospholipid antibody syndrome and enables successful renal transplantation.

Renal transplantation in patients with antiphospholipid antibodies has historically proven challenging due to increased risk for thrombosis and allograft failure. This is especially true for patients with antiphospholipid antibody syndrome (APS) and its rare subtype, the catastrophic antiphospholipid antibody syndrome (CAPS). Since a critical mechanism of thrombosis in APS/CAPS is one mediated by complement activation, we hypothesized that preemptive treatment with the terminal complement inhibitor, eculizumab, would reduce the extent of vascular injury and thrombosis, enabling renal transplantation for patients in whom it would otherwise be contraindicated. Three patients with APS, two with a history of CAPS, were treated with continuous systemic anticoagulation together with eculizumab prior to and following live donor renal transplantation. Two patients were also sensitized to human leukocyte antigens (HLA) and required plasmapheresis for reduction of donor-specific antibodies. After follow-up ranging from 4 months to 4 years, all patients have functioning renal allografts. No systemic thrombotic events or early graft losses were observed. While the appropriate duration of treatment remains to be determined, this case series suggests that complement inhibitors such as eculizumab may prove to be effective in preventing the recurrence of APS after renal transplantation.

International clinical practice guidelines for the treatment and prophylaxis of venous thromboembolism in patients with cancer.

BACKGROUND: Guidelines addressing the management of venous thromboembolism (VTE) in cancer patients are heterogeneous and their implementation has been suboptimal worldwide. OBJECTIVES: To establish a common international consensus addressing practical, clinically relevant questions in this setting. METHODS: An international consensus working group of experts was set up to develop guidelines according to an evidence-based medicine approach, using the GRADE system. RESULTS: For the initial treatment of established VTE: low-molecular-weight heparin (LMWH) is recommended [1B]; fondaparinux and unfractionated heparin (UFH) can be also used [2D]; thrombolysis may only be considered on a case-by-case basis [Best clinical practice (Guidance)]; vena cava filters (VCF) may be considered if contraindication to anticoagulation or pulmonary embolism recurrence under optimal anticoagulation; periodic reassessment of contraindications to anticoagulation is recommended and anticoagulation should be resumed when safe; VCF are not recommended for primary VTE prophylaxis in cancer patients [Guidance]. For the early maintenance (10 days to 3 months) and long-term (beyond 3 months) treatment of established VTE, LMWH for a minimum of 3 months is preferred over vitamin K antagonists (VKA) [1A]; idraparinux is not recommended [2C]; after 3-6 months, LMWH or VKA continuation should be based on individual evaluation of the benefit-risk ratio, tolerability, patient preference and cancer activity [Guidance]. For the treatment of VTE recurrence in cancer patients under anticoagulation, three options can be considered: (i) switch from VKA to LMWH when treated with VKA; (ii) increase in LMWH dose when treated with LMWH, and (iii) VCF insertion [Guidance]. For the prophylaxis of postoperative VTE in surgical cancer patients, use of LMWH o.d. or low dose of UFH t.i.d. is recommended; pharmacological prophylaxis should be started 12-2 h preoperatively and continued for at least 7-10 days; there are no data allowing conclusion that one type of LMWH is superior to another [1A]; there is no evidence to support fondaparinux as an alternative to LMWH [2C]; use of the highest prophylactic dose of LMWH is recommended [1A]; extended prophylaxis (4 weeks) after major laparotomy may be indicated in cancer patients with a high risk of VTE and low risk of bleeding [2B]; the use of LMWH for VTE prevention in cancer patients undergoing laparoscopic surgery may be recommended as for laparotomy [Guidance]; mechanical methods are not recommended as monotherapy except when pharmacological methods are contraindicated [2C]. For the prophylaxis of VTE in hospitalized medical patients with cancer and reduced mobility, we recommend prophylaxis with LMWH, UFH or fondaparinux [1B]; for children and adults with acute lymphocytic leukemia treated with l-asparaginase, depending on local policy and patient characteristics, prophylaxis may be considered in some patients [Guidance]; in patients receiving chemotherapy, prophylaxis is not recommended routinely [1B]; primary pharmacological prophylaxis of VTE may be indicated in patients with locally advanced or metastatic pancreatic [1B] or lung [2B] cancer treated with chemotherapy and having a low risk of bleeding; in patients treated with thalidomide or lenalidomide combined with steroids and/or chemotherapy, VTE prophylaxis is recommended; in this setting, VKA at low or therapeutic doses, LMWH at prophylactic doses and low-dose aspirin have shown similar effects; however, the efficacy of these regimens remains unclear [2C]. Special situations include brain tumors, severe renal failure (CrCl<30 mL min(-1) ), thrombocytopenia and pregnancy. Guidances are provided in these contexts. CONCLUSIONS: Dissemination and implementation of good clinical practice for the management of VTE, the second cause of death in cancer patients, is a major public health priority.

International clinical practice guidelines for the treatment and prophylaxis of thrombosis associated with central venous catheters in patients with cancer.

BACKGROUND: Although long-term indwelling central venous catheters (CVCs) may lead to pulmonary embolism (PE) and loss of the CVC, there is lack of consensus on management of CVC-related thrombosis (CRT) in cancer patients and heterogeneity in clinical practices worldwide. OBJECTIVES: To establish common international Good Clinical Practices Guidelines (GCPG) for the management of CRT in cancer patients. METHODS: An international working group of experts was set up to develop GCPG according to an evidence-based medicine approach, using the GRADE system. RESULTS: For the treatment of established CRT in cancer patients, we found no prospective randomized studies, two non-randomized prospective studies and one retrospective study examining the efficacy and safety of low-molecular-weight heparin (LMWH) plus vitamin K antagonists (VKAs). One retrospective study evaluated the benefit of CVC removal and two small retrospective studies were on thrombolytic drugs. For the treatment of symptomatic CRT, anticoagulant treatment (AC) is recommended for a minimum of 3 months; in this setting, LMWHs are suggested. VKAs can also be used, in the absence of direct comparisons of these two types of anticoagulants in this setting [Guidance]. The CVC can be kept in place if it is functional, well-positioned and non-infected and there is good resolution under close surveillance; whether the CVC is kept or removed, no standard approach in terms of AC duration has been established [Guidance]. For the prophylaxis of CRT in cancer patients, we found six randomized studies investigating the efficacy and safety of VKA vs. placebo or no treatment, one on the efficacy and safety of unfractionnated heparin, six on the value of LMWH, one double-blind randomized and one non randomized study on thrombolytic drugs and six meta-analyses of AC and CVC thromboprophylaxis. Type of catheter (open-ended like the Hickman(®) catheter vs. closed-ended catheter with a valve like the Groshong(®) catheter), its position (above, below or at the junction of the superior vena cava and the right atrium) and method of placement may influence the onset of CRT on the basis of six retrospective trials, four prospective non-randomized trials, three randomized trials and one meta-analysis. In light of these data: use of AC for routine prophylaxis of CRT is not recommended [1A]; a CVC should be inserted on the right side, in the jugular vein, and distal extremity of the CVC should be located at the junction of the superior vena cava and the right atrium [1A]. CONCLUSION: Dissemination and implementation of these international GCPG for the prevention and treatment of CRT in cancer patients at each national level is a major public health priority, needing worldwide collaboration.

Novel analysis of clinically relevant diagnostic errors in point-of-care devices.

BACKGROUND: To ensure proper clinical decision-making and avoid preventable harm, the quality of point-of-care (POC) device measures is routinely assessed. Traditional analyses may not reveal clinically important diagnostic errors. OBJECTIVES: To compare results between a novel analytic framework and traditional analyses. METHODS: Patients in four anticoagulation clinics provided two measures of the International Normalized Ratio (INR) at the same visit as part of routine quality assurance: one via a venous sample and one fingerstick. These were assessed with Hemochron POC devices. Traditional, quarterly, quality assurance assessments emphasized correlation analysis. The novel analysis used enhanced graphics and a validated assessment of clinical decision-making. RESULTS: 1518 paired INRs were analyzed. The correlation between the POC and laboratory assessments ranged between 0.84 and 0.91. Traditional quality assurance showed that the Hemochron devices were acceptable for continued use in each quarterly analysis. Enhanced graphical analysis demonstrated that the Hemochron devices never reported seven common INR values. The Hemochron devices systematically inflated values < 3 and deflated values > 4, biasing results towards the target INR range. Consequently, the Hemochron devices lead to a different clinical decision than the clinical laboratory measure in 31% of cases (458/1466; 95% confidence interval [CI] 29-34). When the reference INR was low, the Hemochron devices would not result in appropriate dose increases in 52% of cases (95% CI 48-56), placing these patients at risk for a significant adverse drug event. CONCLUSIONS: Our novel, clinically relevant analysis revealed previously undetected deficiencies in our POC INR devices, and our approach should be adopted by industry, regulators, and institutions.

Clinical decision-making as the basis for assessing agreement between measures of the International Normalized Ratio.

BACKGROUND: The now classic approach of Bland and Altman is often used to assess the level of agreement between International Normalized Ratio (INR) measures. However, we are concerned that this method does not define agreement in a clinically meaningful way. Agreement between measures should be characterized explicitly in terms of clinical decisions that result from INR measures. OBJECTIVES: To develop and validate an extension of the Bland-Altman method to assess agreement between INR measures, based explicitly on the way clinicians make decisions. METHODS AND RESULTS: We developed a clinically based graphical method to estimate the level of agreement between measures of INR. We identified clinically relevant INR ranges using epidemiologic and clinical evidence regarding risk and expected outcome at different INR ranges. Clinical decisions were expected to agree within these INR ranges and, therefore, the ranges became the basis for establishing agreement between measures. We used paired INR measures and resultant clinical decisions measured during a previous prospective study to validate and compare the accuracy of our model to those of Bland and Altman's and other published models. Our method more accurately predicts when warfarin dosing decisions differ than the Bland-Altman method (P < 0.02). Our method is also superior to other published methods, particularly at the important task of identifying when measures lead to discrepant clinical decisions. CONCLUSIONS: We introduced and validated an improvement of the Bland-Altman method to assess agreement between INR measures. Our model is superior because it is based explicitly on factors that influence clinical decision-making.

Treatment of venous thromboembolism with low-molecular-weight heparin: a synthesis of the evidence published in systematic literature reviews.

OBJECTIVE: To evaluate the methodology and cumulative evidence presented in systematic reviews of clinical trials comparing low-molecular-weight heparin (LMWH) with unfractionated heparin (UFH) for the treatment of venous thromboembolism. METHODS: We reviewed all systematic reviews of clinical trials published until March 2002. Fourteen systematic literature reviews were published between 1994 and 2000. Deficiencies in methodological quality were common, particularly in the description of search strategies, assessment of clinical trial quality, and methods used to combine results. RESULTS: Results of reviews indicate that LMWH is superior to UFH for the treatment of venous thromboembolism, particularly in reducing mortality. Patients with isolated deep venous thrombosis or deep venous thrombosis with concomitant pulmonary embolism seemed to have similar benefit. However, the benefits of LMWH over UFH were smaller in magnitude in reviews that included more recent clinical trials.

A mail survey of United States hematologists and oncologists: a comparison of business reply versus stamped return envelopes.

Mailed surveys are a popular means of obtaining data on large populations. In July 1999 a mail survey was conducted among 3000 randomly selected members of the American Society of Hematology to assess their approach to diagnosis and treatment of polycythemia vera. Because the researchers and the study population are members of the same professional organization with a vested interest in the results, we anticipated that the advantages of return stamped postage seen in previous studies would be less significant. The response rate for stamped return envelopes was 38% versus 32% for business reply envelopes. This statistically significant difference (P =.0005) of six percentage points is comparable to previous research. Excluding labor, the total cost per returned survey was $2.62 for business reply envelopes versus $1.82 for stamped return envelopes. We conclude that stamped return envelopes are a more effective and cost-efficient means of procuring data from physician specialists.

Vena caval filters: a comprehensive review.

Hematologists are often asked to treat patients with venous thromboembolic disease. Although anticoagulation remains the primary therapy for venous thromboembolism, vena caval filters are an important alternative when anticoagulants are contraindicated. To assess the evidence supporting the utility of these devices, a comprehensive review of the English language literature was performed. Except for one randomized trial, the vena caval filter literature consists of case series or consecutive case series. The mean duration of follow-up for each of the 5 filter types varies from 6 to 18 months. All are about equally effective in the prevention of pulmonary embolism (2.6%-3.8%). Deep venous thrombosis (6%-32%) and inferior vena cava thrombosis (3.6%-11.2%) after filter placement vary widely among different filter types primarily because of differences in outcome assessment. Thrombosis at the insertion site is a common complication of filter placement (23%-36%). In view of the absence of randomized comparisons, no filter can be designated as superior in safety or efficacy. Vena caval filters represent a potentially important but poorly evaluated therapeutic modality in the prevention of pulmonary emboli. Randomized trials are necessary to establish the appropriate place for vena caval filters in the treatment of venous thromboembolic disease. (Blood. 2000;95:3669-3677)

Cloning of a human UDP-galactose:2-acetamido-2-deoxy-D-glucose 3beta-galactosyltransferase catalyzing the formation of type 1 chains.

Biochemical evidence suggests that the galactosyltransferase activity synthesizing type 1 carbohydrate chains is separate from the well characterized enzyme that is responsible for the synthesis of type 2 chains. This was recently confirmed by the cloning, from melanoma cells, of an enzyme capable of synthesizing type 1 chains, which was shown to have no homology to other galactosyltransferases. We report here the molecular cloning and functional expression of a second human beta3-galactosyltransferase distinct from the melanoma enzyme. The new beta3-galactosyltransferase has homology to the melanoma enzyme in the putative catalytic domain, but has longer cytoplasmic and stem regions and a carboxyl-terminal extension. Northern blots showed that the new gene is present primarily in brain and heart. When transfected into mammalian cells, this gene directs the synthesis of type 1 chains as determined by a monoclonal antibody specific for sialyl Lewisa. A soluble version of the cloned enzyme was expressed in insect cells and purified. The soluble enzyme readily catalyzes the transfer of galactose to GlcNAc to form Gal(beta1-3)GlcNAc. It also has a minor but distinct transfer activity toward Gal, LacNAc, and lactose, but is inactive toward GalNAc.

The mUBC9 murine ubiquitin conjugating enzyme interacts with the E2A transcription factors.

The ubiquitin-mediated degradation of cellular proteins requires the sequential activity of E1, E2 and, in some cases, E3 enzymes. Using the yeast two-hybrid system, we have cloned 1.0- and 2.5-kb cDNAs encoding the identical murine E2, or ubiquitin conjugating enzyme by virtue of its interaction with the E2A transcription factor. This cDNA encodes the 158-amino-acid protein, mUBC9, which has considerable sequence homology to UBC9 from Saccharomyces cerevisiae and HUS5 from Schizosaccharomyces pombe and is identical to the human UBC9 protein. HUS5 is essential for DNA damage repair, whereas UBC9 is necessary for G2/M progression. The human protein has been shown to correct the UBC9 defect in yeast. Antisera raised against bacterially expressed mUBC9 fusion protein recognize a murine cellular protein of approximately 18 kDa, corresponding to the predicted mobility. Unlike E2A, the mUBC9 protein level is not regulated by serum growth factors. The activity of the apparent homologues UBC9 and HUS5 suggests that mUBC9 may be involved in the degradation of key nuclear proteins that regulate cell cycle progression.

Exploring the substrate specificities of alpha-2,6- and alpha-2,3-sialyltransferases using synthetic acceptor analogues.

The acceptor specificities of rat liver Gal(beta 1-4)GlcNAc alpha-2,6-sialyltransferase, recombinant full-length human liver Gal(beta 1-4)GlcNAc alpha-2,6-sialyltransferase, and a soluble form of recombinant rat liver Gal(beta 1-3/4)GlcNAc alpha-2,3-sialyltransferase were studied with a panel of analogues of the trisaccharide Gal(beta 1-4)GlcNAc(beta 1-2)Man(alpha 1-O)(CH2)7CH3. These analogues contain structural variants of D-galactose, modified at either C3, C4 or C5 by deoxygenation, fluorination, O-methylation, epimerization, or by the introduction of an amino group. In addition, the enantiomer of D-galactose is included. The alpha-2,6-sialyltransferases tolerated most of the modifications at the galactose residue to some extent, whereas the alpha-2,3-sialyltransferase displayed a narrower specificity. Molecular dynamics simulations were performed in order to correlate enzymatic activity to three-dimensional structure. Ineffective acceptors for rat liver alpha-2,6-sialyltransferase were shown to be inhibitory towards the enzyme; likewise, the alpha-2,3-sialyltransferase was found to be inhibited by all non-substrates. Modified sialyloligosaccharides were obtained on a milligram scale by incubation of effective acceptors with one of each of the three enzymes, and characterized by 500-MHz 1H-NMR spectroscopy.

E2A basic-helix-loop-helix transcription factors are negatively regulated by serum growth factors and by the Id3 protein.

Id3, a member of the Id multigene family of dominant negative helix-loop-helix transcription factors, is induced sharply in murine fibroblasts by serum growth factors. To identify relevant targets of Id3 activity, the yeast two-hybrid system was used to identify proteins that dimerize with Id3. Four murine cDNAs were identified in the screen, all of which encode helix-loop-helix proteins: E12, E47, ALF1 and Id4. Co-immunoprecipitation assays confirm that Id3 interacts with E12, E47 and two alternative splice products of ALF1 in vitro. Id3 disrupts DNA binding by these proteins in vitro and blocks transcriptional activation by these factors in cultured murine cells. Additionally, Id3 shows evidence of interacting with the related proteins E2-2 and MyoD, but not c-Myc. These results suggest that Id3 can function as a general negative regulator of the basic-helix-loop-helix family of transcription factors exemplified by the 'E' proteins and MyoD. Although it was previously suspected that E2A is constitutively expressed, our data indicate that E2A is induced in quiescent fibroblasts, by growth factor withdrawal but not by contact inhibition of cell proliferation. These observations extend the role of Id3 in the functional antagonism of E2A-class transcription factors, and suggest that E2A proteins may mediate growth inhibition.

A continuous spectrophotometric assay for glycosyltransferases.

This paper describes a continuous spectrophotometric assay for glycosyltransferases. In this assay, a nucleotide diphosphate is coupled to NADH oxidation via pyruvate kinase and lactate dehydrogenase. The nucleotide diphosphate is produced either directly during the glycosyltransferase mediated reaction, or indirectly by the production of a nucleotide monophosphate during the glycosyltransferase mediated reaction, and subsequent conversion of the nucleotide monophosphate to nucleotide diphosphate using nucleoside monophosphate kinase. Using this assay, kinetic parameters for fucosyl-, sialyl-, and N-acetylglucosaminyltransferases were determined. The assay not only allows continual monitoring of the enzymatic reaction, but is rapid and allows the processing of 96 samples at once since it is performed in 96-well microtiter plates. In addition, the procedure provides a means of monitoring the activity of these enzymes using sugar-nucleotide donor analogs, where radiochemical procedures cannot be used.

Crystallization and preliminary crystallographic investigations of the soluble glucose dehydrogenase from Acinetobacter calcoaceticus.

Single crystals of the soluble glucose dehydrogenase (GDH) from Acinetobacter calcoaceticus have been grown by the vapour diffusion method. These crystals diffract to beyond 2.1 A and are suitable for X-ray crystallography. The space group was determined to be P2(1) with unit cell parameters a = 55.5 A, b = 104.5 A, c = 86.5 A and beta = 99.8 degrees. One asymmetric unit contains a dimer of the GDH molecule.